P810: A NOVEL INDIRUBIN-3-MONOXIME DERIVATIVES FUNCTION AS DISRUPTORS OF PROTEOSTASIS VIA TARGETING TRIM28 IN MULTIPLE MYELOMA
Authors: Teng Fang, Hao Sun, Xiyue Sun, Lixin Gong, Peixia Tang, Zhen Yu, Lanting Liu, Shiyi Xie, Mu Hao, Lugui Qiu
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Despite the availability of several novel therapies, multiple myeloma (MM) remains incurable, making the search for new therapeutic targets and treatments highly significant. Agents disturbing the proteostasis like proteasome inhibitors play indispensable roles in myeloma treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) is a novel proteasome inhibitor that effectively inhibits MM cell proteasome activity. Additionally, research has demonstrated that histone deacetylase 6 (HDAC6) inhibitors could inhibit the aggresome-autophagy pathway. Therefore, a new I3MO derivative, 8b, was synthesized by coupling an HDAC6 inhibitor to the I3MO structure. Aims: We aimed to investigate the cytotoxic effect of 8b on multiple myeloma and elucidate its regulatory role in the ubiquitin-proteasome system and aggresome-autophagy system in MM cells. Additionally, we aimed to identify potential targets of 8b and its mechanism of action. Methods: 8b effects on myeloma treatment were investigated via in vivo and in vitro studies. Cell viability and flow cytometry were performed for evaluating myeloma cytotoxicity. Western blot, immunofluorescence, and proteasome activity assay were performed to investigate the effect of 8b on MM cell proteasome activity, aggresome formation, and autophagosome formation. Dual-luciferase reporter gene assay was used to identify novel downstream targets of 8b. The role of TRIM28 was further assessed in shRNA knock-down (KD) experiments. Results: We successfully identified a novel I3MO derivative, 8b, which could dose-and time-dependently inhibit myeloma cell proliferation and induce apoptosis. Exposure to 8b was associated with the accumulation of cells in S phase as well. Our in vivo studies also showed that compound 8b repressed myeloma growth. Treatment with 8b led to decreased proteasome activity and inhibited the formation of protein aggresomes and autophagosomes. Additionally, compound 8b could, in combination with bortezomib (BTZ), enhance MM cell apoptosis and potentially overcome BTZ drug resistance. Mechanistically, Dual-luciferase reporter gene assay showed that 8b could downregulate TRIM28 promoter activity, leading to transcriptional repression of TRIM28. Bioinformatic analysis demonstrated that the expression of TRIM28 was positively correlated with the expression of multiple genes encoding proteasome subunits including PSMB1 and PSMB2. Chip-qPCR also showed that TRIM28 could bind with the promoter region of PSMB1 and PSMB2, while knockdown of TRIM28 caused the downregulated expression of PSMB1 and PSMB2, and decreased proteasome activity, which sensitizes MM cells to bortezomib. Additionally, Knockdown of TRIM28 inhibited the formation of autophagosomes, which could also contribute to higher sensitivity to BTZ in MM cells. Summary/Conclusion: We identified a novel I3MO derivative, 8b, which could directly kill myeloma cells while acting synergistically with bortezomib. This represents a novel and promising therapeutic approach that could help overcome the current drug resistance in myeloma. Additionally, we for the first time provided novel important insights into the role of TRIM28 in MM pathobiology, demonstrating that its deregulation strongly correlates with proteostasis, often associated with inferior outcomes of the disease, which should be considered as a potential therapeutic target for MM treatment.Keywords: Multiple myeloma, Drug resistance